Tritiated Opiate Receptor Radioligands

Synthesis and Characterization: Past, Present, and Future Practices

The alleviation of pain is perhaps the greatest long-standing goal of both ancient and modern medicine. Quite recently, opiate receptors were implicated in the propagation of pain. With exquisite complimentary molecular architecture, these protein pockets tightly bind smaller endogenous molecules, setting off a cascade of intracellular events, resulting in the pain response.

This critical realization has spawned a successful strategy for pain relief; namely, that competitive blocking of opiate receptors with even more tightly bound synthetic analgesics (termed opiate antagonists) could successfully disrupt the sensation of pain.

To assist in the identification and optimization of novel and safer analgesics, the past several decades have witnessed the creation of innovative biochemical methods.

Many of the reagent tools PerkinElmer has created for drug discovery have been radioactively tagged small molecules (radioligands). They accurately and sensitively track radioactivity and consequently aid in the understanding of the interaction of these radioligands in cells associated with the biochemistry of pain. Tritium, the radioactive isotope of hydrogen, confers no structural change on the molecule and has thus been an especially useful label.

In 1973, Professor Solomon Snyder of Johns Hopkins University reported a profound technical breakthrough in the general area of drug discovery and the analgesic field in particular. Using the well-known opiate antagonist naloxone, labeled with tritium by PerkinElmer, Professor Snyder identified an opiate receptor through an efficient sensitive biochemical method that has come to be known as a receptor binding assay.

Since then, the receptor binding assay has become a valuable tool for drug discovery, and PerkinElmer has provided optimized radioactive reagents for this technology. The tritium-labeled naloxone first employed by Professor Snyder was indeed radioactive, but the degree of radioactivity incorporation in the molecule was rather low. It was quickly appreciated that higher specific activity opiate receptor radioligands would be needed to increase the assay’s sensitivity and scope, and that new tritiation methods would be required to obtain that goal.

One of the strategies we turned to was catalytic tritium dehalogenation, relying on the ability of a heterogeneous catalyst (usually palladium on some support) to efficiently replace halogens (like bromine and iodine) with tritium atoms in precursor molecules at high specific activity.

Using this technique, we have been able to tritiate many useful opiate receptor (and related) ligands including the kappa opiate receptor agonist bremazocine, the fluorine-containing analgesics brifentanil and ocfentanil, and the nonpeptide substance P antagonist CP-96,345 in close collaboration with Pfizer scientists.

Another way to achieve high specific activity tritiation is by the design and synthesis of unsaturated precursor molecules that could also be catalytically tritiated, providing the desired radioligand at even higher specific activity than possible with catalytic tritium dehalogenation. Indeed, it was by using this technique that we soon revisited naloxone (Figure 1) and tritium-labeled it at far higher specific activity, employing a Lindlar catalyst reduction of a synthetic alkyne precursor.

Furthermore, this new and higher specific activity version of [3H] naloxone (Figure 1) had tritium attached in a specific and far more stable location than that previously employed by Professor Snyder in his early work. Utilizing this improved radioligand, we were able to also synthesize the irreversible dimer analogue [3H] naloxonazine (Figure 2) by means of exacting small-scale radiochemistry. Another example of successful implementation of this radiolabeling method was the tritium labeling of lofentanil, an extremely potent analgesic, with partners at Janssen. Using this technology, PerkinElmer has tritiated literally scores of valuable analgesic compounds at high specific activity.

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